Apparatus and method for processing biological material

ABSTRACT

The application discloses an apparatus and method for processing biological material, including a suspension of cells.

TECHNICAL FIELD

The present subject matter generally relates to an apparatus and methodfor processing biological material to concentrate and wash a biologicalcomponent in the material.

BACKGROUND

Biological materials, such as cells, are used in numerous therapeutic,diagnostic and research applications. For example, stem cells may beadministered to patients to obtain a desired therapeutic effect such asregeneration of tissue in vivo. In other situations, biologicalmaterials including cells may be administered for grafts, transplants,or other procedures.

To provide an effective preparation of the biological material, havingsufficient concentration that may be administered to a patient or thatmay be useful for diagnostic and research purposes, it often isnecessary to perform numerous and lengthy manipulations involving thematerial. For example, stem cells often are first separated and isolatedfrom a tissue from which they are derived, such as muscle, blood oradipose (fat) tissue. The cells of such a composition then may have tobe subjected to multiple rounds of purification, washing or othertreatments before they can be introduced, such as by injection, into apatient. These procedures may require sequential transfer of the cellsto different containers. They also may require further manipulations,such as to promote sedimentation. Each procedure preferably is performedaseptically or in a closed sterile system to limit or avoid thepotential introduction of contaminating material or organisms into thecomposition. Alternatively, even if the cells will not be administeredto a patient but, instead cultured in vitro, for example, they still mayrequire extensive washing and concentration preferably in asepticconditions.

Also, to be suitable for administration to a patient, it may bepreferable for a preparation of biological material to be highlyconcentrated. This may permit a relatively small volume to beadministered. For example, stem cell preparations of about 1×10⁸ cellsor more generally may be concentrated into a volume of less than five(5) mls for injection into a patient.

Although much work has been done in the field of tissue processing,there continues to be a need for advances in the field of processingbiological material including in the areas of washing and concentratingmaterial for subsequent therapeutic, diagnostic, research or otherapplications.

SUMMARY

In one example, the subject matter of this application is directed to asedimentation assembly for concentrating cells in a suspension. Thesedimentation assembly includes a first chamber for receiving thesuspension including a cell population. The first chamber has a cellconcentration zone for receiving a concentrated population of the cellsupon application of a sedimentation force upon the chamber. The assemblyalso includes a second chamber that is adapted to be removably placed influid communication with a fluid destination or source, including theconcentration zone of the first chamber. The first and second chambersas a unit are placeable in a sedimentation force field with the firstand second chambers in fluid communication for flowing a portion of thesuspension including a cell population into the second chamber. Thechambers are preferably physically separable so that fluid communicationis effected physically by joining the chambers or broken by physicallyseparating the chambers.

In another example, the disclosed subject matter is directed to asedimentation assembly for washing and concentrating a cell populationin a suspension. The sedimentation assembly includes a first chamber forreceiving a suspension including a cell population. The sedimentationassembly also includes a second chamber, adapted to be removably placedin fluid communication with a fluid destination or source, including thefirst chamber. The first and second chambers are placeable as a unit ina sedimentation force field with the first and second chambers in fluidcommunication, such that when the unit is subjected to the sedimentationforce field at least a portion of the suspension flows from the firstchamber to the second chamber, thereby forming a concentrated cellsuspension in the second chamber.

The disclosure also is directed to methods of concentrating cells in asuspension. In one example, a method of concentrating cells in asuspension includes collecting a suspension including a cell populationwithin a first chamber. The cell population is sedimented to obtain aconcentrated cell suspension within the first chamber and theconcentrated cell suspension is flowed into a second chamber under asedimentation force field.

In a further example, a method of concentrating and washing cells in asuspension is disclosed. The method includes collecting a suspensionincluding a cell population within a first chamber and sedimenting thecell population to obtain a concentrated cell suspension within thefirst chamber. The concentrated cell suspension is flowed into a secondchamber under a sedimentation force field. The second chamber isdetached from the first chamber and the concentrated cell suspension isflowed into a further fluid destination or source. The further fluiddestination or source is placeable together with the second chamber in asedimentation force field.

In a further example, an apparatus for reconstituting, washing ortreating a cell preparation is described. The apparatus has a firstchamber with at least one port. The apparatus also includes a secondchamber that has at least one port and that is adapted to be repeatedlyand removably placed in fluid communication with a fluid destination orsource, such as the first chamber. At least one port of the firstchamber has a resealable valve and at least one port of the secondchamber has a member for opening the valve.

A method for reconstituting, washing or treating a cell preparation isalso disclosed. The method includes placing a cell preparation within afirst chamber and flowing the cell preparation from the first chamberinto a second chamber which is adapted to be repeatedly and removablyconnected to and placed in fluid communication with the first chamber.One of the first and second chambers has a port having an automaticallyresealable valve and the other of the first and second chambers has aport having a member adapted to automatically open the valve when thechambers are connected. The second chamber is then disconnected from thefirst chamber and the valve automatically closed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partial cross-sectional view of one example of asedimentation assembly according to the disclosure where first andsecond chambers are shown in a separated position and out of fluidcommunication;

FIG. 1 a is an enlarged cross-sectional view of one example of acoupling between the first and second chambers of FIG. 1, with thechambers shown in a separated position;

FIG. 2 is a partial cross-sectional view of the of sedimentationassembly of FIG. 1 with the first and second chambers shown in aconnected position in fluid communication.

FIG. 2 a is an enlarged cross-sectional view of the example of acoupling between the first and second chambers of FIG. 2, with thechambers shown in a connected position;

FIGS. 3 a-3 f show one example of a method of using the sedimentationassembly of FIG. 1 according to the disclosure;

FIG. 4 is a perspective view of one example of a holder, holding amodified sedimentation assembly for use in a sedimentation force field,specifically generated by a centrifuge;

FIG. 5 shows a further example of a sedimentation assembly with aholder, such as the holder of FIG. 4;

FIG. 6 is a cross-sectional view of the example of the holder with thesedimentation assembly of FIG. 4 located in the holder;

FIGS. 7 a-7 g show an example of a method of using the sedimentationassembly of FIG. 1 according to the disclosure;

FIGS. 8 a-8 h show an example of a method of use of anothersedimentation assembly according to the disclosure, where one chamberincludes a plunger;

FIG. 9 is a cross-sectional view of a further example of a sedimentationassembly according to the disclosure.

FIGS. 10 a-d are cross-sectional views of further examples of valves andconnectors that may be used with an apparatus disclosed herein.

DETAILED DESCRIPTION

While detailed examples are disclosed herein, it is to be understoodthat these disclosed examples are merely exemplary, and various aspectsand features described herein may have utility alone or in combinationwith other features or aspects in a manner other than explicitly shownbut would be apparent to a person of ordinary skill in the art.

The subject matter of the present application is directed generally toan apparatus and method for processing biological material. In oneexample, the apparatus is a sedimentation assembly that may be used toconcentrate biological material. In other preferred examples, thesedimentation assembly may be used to reconstitute, wash and/orotherwise treat the material with desired reagents and solutions. Forexample, the apparatus may be used to wash or treat cell preparationswith selected buffers. In other examples, the apparatus may be used totreat a cell preparation with reagents such as serum, antibodies orgrowth factors. In further examples, the apparatus may be used toprepare cells for freezing and storage and may be used reconstitute acell preparation that had been frozen and which may be required to betransferred to culture media.

In other preferred examples, the apparatus may be used to reconstitute,wash or otherwise treat a preparation of cells without necessarilysedimenting the cells. For example, the apparatus may be used totransfer a thawed cell preparation to tissue culture media so that thecells may be cultured.

Turning to the accompanying drawings, FIG. 1 illustrates a sedimentationassembly generally at 10 that may be used in concentrating biologicalmaterial, such as cells, from tissue. The sedimentation assemblyincludes a first chamber 12 that may receive biological material, suchas a suspension of cells. The sedimentation assembly 10 also includes asecond chamber 26 that may be placed in fluid communication with thefirst chamber 12, for example, as seen in FIG. 2. That is, the firstchamber 12 and second chamber 26 may be readily coupled together orconnected to form a sedimentation assembly 10 as a stable, integratedunit. The chambers 12, 26 then may be separated and then reconnected, ifnecessary, so that fluid communication between the chambers may berepeatedly established, removed and re-established. For example, FIG. 1shows the sedimentation assembly 10 with the first and second chambers12, 26 separated—and thus fluid communication has not yet beenestablished or has been removed. FIG. 2 shows the assembly 10 with thetwo chambers connected or having been reconnected and placed in fluidcommunication. As shown in FIGS. 1 and 2, a coupling 32 may be used tofacilitate the connection, separation and reconnection of the twochambers.

In one example, the first chamber 12 is substantially rigid and thesecond chamber 26 may have the same or different degree of rigidity. Thechambers, for example, may be generally be more rigid than bags commonlyused in blood processing procedures, but may retain a degree offlexibility. Thus, in some examples, the chambers may be sufficientlypliable such that they may be manipulated by the application of no morethan an average manual force. The chambers 12, 26 may be formed, atleast in part, of substantially rigid transparent plastics such that thecontents may be viewed during processing. Of course, the first andsecond chambers need not necessarily be made of the same materials orhave the same degree of rigidity. In one preferred example, at leastpart of the second chamber 26 may be less rigid than the first chamber12, thereby permitting the volume of the second chamber to bemanipulated or expelled by the application of force to the wall of thesecond chamber or by a change in pressure of the chamber.

The sedimentation assembly also is preferably disposable, and may bemade from polyethylene, polypropylene or other materials that aresuitable for use with biological material and that may be easilysterilized before use, or otherwise provided in a sterile form. Althoughtypically not believed to be necessary, the chamber surfaces may betreated or coated with materials such as serum, albumin, polycations,polyanions, or other materials, as desired, using methods known in theart, to increase or decrease the adherence or affinity of selectedbiological material to the walls of the first and second chambers, orfor other purposes.

The volumes of the first and second chambers 12, 26 may be selecteddepending on particular requirements. In one example, such as shown inFIG. 1, the second chamber 26 has a smaller volume than the firstchamber 12. This example may be used, for example, when the suspensionof cells is to be concentrated into a smaller volume for administrationto a patient or for further processing. The chambers 12, 26 also mayassume numerous shapes, as desired. For example, as described furtherherein, one or both chambers may be in the form of a syringe with amoveable plunger therein.

In the example shown in FIG. 1, the first chamber 12 has an upper wallportion 14 which is cylindrical. The upper wall portion 14 of the firstchamber 12 is closed at an upper end by a wall or base 15 and is joinedat a lower end to a conical or tapered portion, forming a concentrationzone or area 16 within the first chamber 12, proximate its lower end. Asshown in FIG. 1, an inlet tubing 20 may be attached to the first chamber12 via an aperture 18 in the base 15. The inlet tubing 20 may be used tointroduce biological material including a suspension of cells into thefirst chamber 12. The first chamber 12 also has an outlet 22 adjacentthe lower end of the concentration zone 16. The first chamber 12 furtherincludes a vent 24 in the base 15 to permit venting of air as may berequired when fluid is being added to or removed from the first chamber12.

In the example shown in FIGS. 1 and 2, the second chamber 26 is shown ashaving a substantially rigid spherical shape with a port 28 to permitthe introduction and/or removal of fluid. Of course, the second chamber26 may be constructed to be more or less flexible and to have adifferent shape, as desired. In this example, the second chamber 26 alsoincludes a lower pocket or region 30 opposite the port 28. The pocket 30provides a space or zone where cells can accumulate duringsedimentation, and may facilitate later removal of a fluid from thesecond chamber 26 with less disruption to the cells collected in thepocket 30. Of course, the sedimented cells may be suspended within thesecond chamber and used directly as a final suspension for a desiredpurpose such as injection into a patient without further processing.

As noted above, in FIG. 1, the second chamber 26 is shown as physicallyseparated from the first chamber 12. Therefore, the second chamber 26has not yet established or has been removed from fluid communicationwith the first chamber 12. FIG. 2 shows the second chamber 26 asconnected to the first chamber 12, so that the second chamber 26 isplaced in fluid communication with the first chamber 12.

As shown in FIGS. 1 and 2, a separable coupling 32 may be utilized tofacilitate the connection, separation and reconnection of the firstchamber 12 and second chamber 26. FIGS. 1 a and 2 a showcross-sectional, enlarged views of an example coupling 32. FIG. 1 ashows an arrangement of the coupling when the chambers 12, 26 are notconnected and not in fluid communication with each other. FIG. 2 a showsan arrangement when the chambers 12, 26 are connected and fluidcommunication between the chambers may have been established.

As shown in FIGS. 1 a and 2 a, the illustrated coupling 32 includes twomating elements. A first mating connector or element 34 of the coupling32 is shown as being externally threaded at its upper end, and engagedwith the first chamber 12 via complementary threads in the outlet 22. Itwill be appreciated that the first mating element 34 may be constructedin other ways to engage the first chamber 12 or may be molded with orotherwise connected to the first chamber 12. The first element 34 shownin FIGS. 1 a and 2 a also includes an outer collar 35 that is internallythreaded, a blunt cannula 36, located within the collar.

A second mating connector or element 38 of the coupling 32 may bethreaded, molded or otherwise connected to the second chamber 26 at itsport 28. In the example illustrated in FIG. 1 a, the second matingelement 38 is shown with internal threads at its lower end that engagecomplementary external threads extending from the port 28 at the top ofthe second chamber 26. The second mating element 38 also includes at itsupper end an external thread or flange 37 for mating with the internallythreaded collar 35 of the first mating element 34.

In this illustrated example, the second mating element 38 of thecoupling 32 further includes a flexible pre-slit, re-sealable septumvalve 40. As seen in FIG. 1 a, the septum valve 40 is biased towards aclosed position. Therefore, the septum valve 40 automatically closes andseals the second chamber 26 from the environment when the first andsecond chambers 12, 26 are separated. As seen in FIG. 2 a, the septumvalve 40 also automatically seals against the cannula 36 when thechambers 12, 26 are connected.

The disclosed apparatus is not limited to a particular connector orvalve construction shown. For example, the above elements may beotherwise constructed or reversed in their placement, if desired. Italso will be appreciated that other examples may include valves on bothchambers, as desired.

To join the two chambers 12, 26 and place them in fluid communication,the first and second mating elements 34, 38 of the coupling 32 areconnected together. This causes the cannula 36 to pass through there-sealable septum valve 40, as indicated in FIG. 2 a. In thisarrangement, the connector provides a closed passageway or channel 42 inthe sedimentation assembly 10 that is sealed from the environment. Inthis regard, the septum valve is preferably elastically stretched aboutthe penetrating member. In this example with the first and secondchambers 12, 26 connected as a unit, fluid including cells i.e a cellsuspension (or liquid alone), may flow in either direction (firstchamber 12 to second chamber 26 or second chamber 26 to first chamber12) depending on the direction and magnitude of forces applied to thesedimentation assembly 10. To remove the fluid communication between thechambers 12, 26, the cannula 36 is withdrawn from the septum valve 40,which automatically re-seals instantaneously.

FIGS. 3 a-3 f illustrates generally a method of use of a sedimentationassembly 10. As shown in FIGS. 3 a and 3 b, the first chamber 12, whichhas received a suspension of cells, may be connected to a second chamber26 and fluid communication between the chambers may be established. Acoupling 32 may be used to facilitate the connection of the twochambers, creating a sedimentation assembly 10 in the form of anintegrated unit, with the chambers 12, 26 rigidly connected together bythe coupling 32, as seen in FIG. 3 b.

The sedimentation assembly 10 may be placed in a sedimentation forcefield, such as a centrifugal force field, although a simplegravitational force field, i.e. normal gravitational force, may besufficient to promote sedimentation in certain circumstances. Thesedimentation force field, such as developed by centrifugation in FIG. 3c, should be sufficient to cause desired cells of the suspension tobecome concentrated in the concentration zone 16 of the first chamber 12and, optionally, to flow from the first chamber 12 to the second chamber26.

After the second chamber 26 receives a quantity of the desiredsuspension of cells, the second chamber 26 may be separated from thefirst chamber 12, as illustrated in FIGS. 3 d and 3 e. Thus, thesedimentation assembly 10 may be inverted, as shown in FIG. 3 d, toreduce potential spillage as the cannula 36 is removed from the septumvalve 40. The second chamber 26 then may be disconnected at the coupling32 from the first chamber 12, such as by disengaging the internalthreads of the collar 35 from the flange 37 on the second chamber 26,and withdrawing the cannula 36.

With the second chamber 26 disconnected and separated from the firstchamber 12, as indicated in FIG. 3 f, the concentrated suspension ofcells may be removed from the second chamber 26 such as by use of asyringe 41. If desired, the cells also may be maintained in the secondchamber 26, such as for further processing. For example, the separatedsecond chamber 26 with the desired cells may be placed in fluidcommunication with a further fluid destination or source, such as anadditional chamber, for further treatment and concentration, asdescribed below in reference to another example.

The example sedimentation assembly 10 may be used to reconstitute, wash,treat or concentrate a diverse set of cell preparations. For example,the biological material received by the first chamber 12 may be arelatively crude suspension of cells and may include individual cells,multi-cellular aggregates and/or cells associated with non-cellularmaterial. The suspension of cells may include one or more cell types.The suspension of cells also may include stem cells alone or incombination with other cell types, including other types of stem cells.

The sedimentation assembly 10 also may be used with cell preparationsthat have been subjected to purification procedures. For example, thesedimentation assembly 10 may be linked, connected to or otherwiseincorporated into a system for purifying cells. In such an arrangement,the first chamber 12 of the sedimentation assembly 10 may receive asuspension of cells from the cell purification system. For instance, thesuspension of cells received by the first chamber may be stem cells thathave been isolated according to the presence or absence of a selectedcell marker using affinity techniques. The suspension of cells may havebeen, for example, isolated as being CD34 positive.

As indicated, centrifugation may be used to produce a sedimentationforce field to flow a suspension of cells from the first chamber 12 tothe second chamber 26. When centrifugation is used, the sedimentationassembly 10 may be placed in a holder, for convenient further placementof the assembly in a centrifuge. The holder also may assist instabilizing the assembly during centrifugation. The size and shape ofthe holder may be adapted to a given sedimentation assembly andcentrifuge bucket. Such a holder also may be used to hold asedimentation assembly for sedimentation at normal gravity force.

FIGS. 4-6 show an example of a holder 44 that may be used with a furtherexample of a sedimentation assembly 48. FIG. 4 shows the example of aholder 44 that may be used to hold a sedimentation assembly 48 in acentrifuge bucket during centrifugation. The holder includes an opening46, best seen in FIG. 5, for placement of the sedimentation assemblyinto the holder 44. In this example, the overall shape of the holdergenerally is cylindrical, to fit the most common shape of centrifugebuckets.

FIG. 5 shows the placement of the sedimentation assembly 48 into theholder 44 of FIG. 4. As shown, the sedimentation assembly includes afirst chamber 50 with a concentration zone, 52 a second chamber 54, anda coupling 56. In this example, the first chamber 50 includes an inlet58 for receiving a suspension of cells. The inlet 58 may be covered, forexample, with a screw cap 60.

In FIG. 6, the sedimentation assembly 48 is shown placed within theholder 44, shown in cross-section, for use in a sedimenting procedure,as would occur during centrifugation. During the sedimenting procedure,the desired cells, initially in the first chamber 50, will becomeconcentrated within the concentration zone 52, and will tend to flowinto the second chamber 54, via the coupling 56.

FIGS. 7 a-7 g exemplify a use of a sedimentation assembly 61 accordingto the disclosure for performing multiple washing and/or treating stepsof a cell population. The sedimentation assembly 61 includes a firstchamber 64 and a second chamber 26. In FIG. 7 a, the second chamber 26contains a suspension of cells 62 that may require further processing.The suspension of cells in the second chamber 26 may result fromprocessing according to previously described examples for obtaining aconcentrated cell population such as is discussed, for example, withrespect to use of the first chamber 12 in FIGS. 3 a-3 f.

As shown in FIG. 7 b, the second chamber 26 with the suspension of cells62 may be placed in fluid communication with another fluid destinationor source, such as an additional first chamber 64 which may contain awashing or treatment solution. The connection of the two chambers may befacilitated by the presence of a coupling, such as previously discussedcoupling 32 that allows for repeated coupling (in fluid communication)and uncoupling (not in fluid communication) of the chambers. The cells62 then may flow into the additional first chamber 64, with the flowbeing enhanced simply by applying manual force to a wall of the secondchamber 26, such as by squeezing the second chamber 26 while thesedimentation assembly 61 is in an inverted position. It will beappreciated that a sedimentation force field, such as a centrifugalforce field, also may be applied to the inverted sedimentation assemblyso as to facilitate the flow of cells from the second chamber 26 to thefirst chamber 64.

In examples where the cells are to be washed, the suspension of cellsmay be flowed from the second chamber 26 to an additional first chamber64 that contains a large volume of a wash solution. In other examples,the cells may be flowed into an additional first chamber containing arelatively small volume of fluid, as might occur when the cells are tobe treated with an expensive reagent. After flowing the cells from thesecond chamber 26 to the additional first chamber 64, to limit cell lossthe second chamber 26 may remain connected with the first chamber 64, oralternatively may be disconnected from the first chamber 64.

After washing or treatment of the cells within the additional firstchamber 64, the cells may be flowed back into the second chamber 26,which remains attached to the additional first chamber thereby allowingcomplete recovery of all the cells or at least reducing cell loss. Thismay be accomplished using a sedimentation force field, such as shown inFIG. 7 c. Alternatively, the additional first chamber 64 may beconnected to and placed in fluid communication with a new secondchamber. The second chamber 26 then may be separated from the additionalfirst chamber 64, resulting in a suspension of cells in the secondchamber 26 that has been washed and re-concentrated, as seen in FIG. 7d.

If desired, the washed suspension of cells in the further second chamber26 then may be flowed to yet another first chamber 68 for furtherprocessing, such as by additional washing or treatment. The connectionand flowing of the suspension of cells from the second chamber 26 to theadditional first chamber 68 is represented in FIG. 7 e and isaccomplished in a similar manner as with respect to the abovedescription of FIG. 7 b. As shown in FIG. 7 f, the cells then may beflowed back to the original second chamber 26 or a new second chamber,such as by use of a sedimentation force field. The first and secondchambers may remain attached and the use of the same second chamber mayreduce cell loss. In this way, a suspension of cells may be repeatedlymoved between “first” and “second” chambers that are placed in fluidcommunication, providing for repeated washing, treatment and/orre-concentration of the cells, shown deposited in the second chamber 26in FIG. 7 g.

FIGS. 8 a-8 h shows a further example of a sedimentation assembly 70 anda method of use thereof in accordance with the disclosure. Thesedimentation assembly 70 includes a first chamber 72 for receiving acell suspension and a second chamber 76, which can be in the form of asyringe. A coupling 78 can be used to place the chambers 72, 76 in fluidcommunication. As described with respect to the other examples, thesecond chamber 76 may be placed in fluid communication with a firstchamber 72. The sedimentation assembly 70 with the first chamber 72connected to the second chamber 76 may be placed in a sedimentationforce field, such as shown in FIG. 8 b, to flow a cell population 74into the second chamber 76.

The flow of the cell population 74 to the second chamber 76, in the formof a syringe, also may be facilitated or accomplished by moving a piston80 of the syringe 76, so as to create a vacuum in the second chamber 76,as shown by the displacement of the piston 80 in FIGS. 8 c and 8 d. Thismovement of the piston 80 causes fluid to be drawn into the secondchamber 76 from the first chamber 72 to relieve the vacuum. The volumeof the syringe chamber may be configured as fixed or variable, dependingon anticipated fluid volume. In one example, retraction of the piston 80will draw fluid into the second chamber thereby helping to recover cellsthat remain in the first chamber 72 or in the area of the coupling 78even after the application of a sedimentation force field. In addition,retraction of the piston may be used to increase the amount of fluid inthe second chamber, if desired. The piston 80 of the syringe 76 also maybe pushed after the cell population has been flowed into the syringe 76,thereby removing excess supernatant from the second chamber andadjusting the volume in which the cells are suspended in the secondchamber 76.

The second chamber 76 then may be removed from fluid communication withthe first chamber 72, as illustrated in FIG. 8 e. Given that the secondchamber 76 is in the form of a syringe, the second chamber 76 may beused to administer the cells to a patient or used for other purposes. Asindicated in FIG. 8 f, the syringe also may be placed in fluidcommunication with a further fluid destination or source, such as afurther first chamber 82, for further washing or treatment. The cells 74may be flowed into the further first chamber 82 by movement of thepiston 80 of the second chamber syringe 76, as shown in FIGS. 8 f and 8g, or by application of a sedimentation force field, such as describedabove in reference to FIG. 8 b. The cells also may be flowed back intothe second chamber 76 (or into a further “second” chamber) to result ina concentrated cell population in the second chamber 76, as shown inFIG. 8 h.

A further example of a sedimentation assembly according to thedisclosure is shown in FIG. 9. According to this example, one or bothchambers of the sedimentation assembly is adapted by the provision ofone or more air pockets to more easily allow the trapping of air in thechamber. This feature is beneficial when it is necessary to easilycompress the contents of a chamber, such as occurs, for example, when aa structure such as needle or cannula must be introduced into a chamberfilled with liquid.

The sedimentation assembly 84 shown in FIG. 9 is substantially similarto the example shown in FIGS. 1 and 2. That is, the sedimentationassembly 84 includes a first chamber 86, a second chamber 88, and acoupling 90. The coupling 90 shown in FIG. 9 is identical to that shownin FIG. 1 a. In FIG. 9, the wall of the second chamber 88 curves upwardson both sides of inlet port 92, forming air-trapping pockets or regions94 within the second chamber 88.

According to the example of FIG. 9, air is trapped in the air-trappingregions 94 when the chamber is placed upright and filled with liquid.When a syringe needle or similar device is inserted into the secondchamber 88 through, for example, the septum 96, liquid is forced intothe air-trapping regions because the trapped air is compressible,allowing a structure such as needle or cannula to more easily penetratethe chamber.

Further, other types of valves and couplings may be used with thesedimentation assembly of the disclosure. Resealable valves arepreferred (and particularly preferably automatically resealable) toregulate the flow of fluid between the chambers, either alone or incombination with other valves. For example, stopcock valves as well asclamps are examples of manually resealable elements that may be used. Inone example, a syringe-type needle may be used with a rubber plugforming a valve.

Other valves and couplings that may be used are disclosed, for example,in U.S. Pat. Nos. 4,683,916, 5,188,620, 5,957,898, 6,039,302 6,261,282and 6,605,076 which are herein incorporated by reference in theirentirety. These valves and others may employ a variety of septums andseptum opening mechanisms, and may be employed with various types andshapes of coupling members such as needles, Luer members, cannulas,nozzles and hybrid structures.

FIG. 10 a-d shows examples of such valves and connectors. In FIG. 10 a,valve 100 has a resealable pre-slit septum 102 mounted on the first end104 of a housing 106. The septum is mounted between annular, U-shaped,swaged end members 108 and an internal septum supporting ridge 110. Asdescribed more fully in U.S. Pat. Nos. 5,188,620 and 6,605,076, thisseptum is co-operative with a blunt cannula that may be inserted throughseptum slit 102 for introducing fluid into and through the valve.

A further example of a valve connector 200 is shown in FIG. 10 b. Inthis example, a nozzle 202 in the form of a male Luer fitting is shownpartially inserted into the valve 200 to establish a fluid flow path.Briefly, the insertion of the nozzle 202 depresses a gland orelastomeric member 204 and axially displaces a hollow internal post 206to open a fluid flow path through the gland and the hollow post to valveoutlet 208.

FIG. 10 c shows a further example of a valve connector that may be usedwith an apparatus according to the disclosure. The valve connector 300includes a resealable valve member 302 having an upper portion 304,middle portion 306 and annular skirt (not shown). One valve slit 308,extends downwardly through the upper portion 304 and middle portion 306into a chamber 310. Engagement of a cannula against the face of thevalve 302 causes the slit 314 to open and provides a fluid flow paththrough the slit and chamber 310 to the valve outlet.

FIG. 10 d shows one further valve that may be used with the presentapparatus. Specifically, the valve body 400 of FIG. 10 d includes a maleLuer portion 402 and a female Luer portion 404. A valve disc 406 islocated within the valve body and rests on a triangular projection 408.The inherent resiliency of the valve disc normally biases it in a closedposition as shown in solid lines. A valve actuator 410 is located in thefemale Luer bore, so that the insertion of a connecting male Luer forcesthe actuator 410 axially to engage and bend the edges of the valve disc406 downwardly to an open position. The disc reseals upon removal of theconnecting male Luer.

It will be understood that the examples provided in the presentdisclosure are illustrative of some of the applications of theprinciples of the present disclosure. Numerous modifications may be madeby those skilled in the art without departing from the true spirit andscope of the disclosure. Various features which are described herein canbe used in any combination and are not limited to particularcombinations that are specifically described herein.

1.-23. (canceled)
 24. A method of concentrating cells in a suspension,comprising: a. collecting a suspension including a cell populationwithin a first chamber; b. sedimenting the cell population to obtain aconcentrated cell suspension within the first chamber; and c. flowingthe concentrated cell suspension into a second chamber in fluid flowconnection with the first chamber under a sedimentation force field. 25.The method of claim 24 further comprising disconnecting the firstchamber from the second chamber and connecting the second chamber to athird chamber for further processing of the concentrated cellsuspension.
 26. The method of claim 24 further comprising disconnectingthe first chamber from the second chamber, removing any contentsremaining in the first chamber, adding a solution to the first chamber,adding the concentrated cell suspension from the second chamber to thefirst chamber and reconnecting the first and second chambers for furtherprocessing.
 27. The method of claim 24 wherein the steps of a-c arerepeated but the suspension including a cell population that iscollected in the first chamber when repeated is the concentrated cellsuspension that was flowed to the second chamber.
 28. A method ofconcentrating or washing cells in a suspension, comprising: a.collecting a suspension including a cell population within a firstchamber; b. sedimenting the cell population to obtain a concentratedcell suspension within the first chamber; c. flowing the concentratedcell suspension into a second chamber in fluid flow connection with thefirst chamber under a sedimentation force field; d. disconnecting thesecond chamber from the first chamber; and e. flowing the concentratedcell suspension into a further fluid destination or source, the furtherfluid destination or source adapted to be placed together with thesecond chamber in a sedimentation force field.
 29. The method of claim28 wherein the sedimentation force field is a centrifugal force field.30. (canceled)
 31. The method of claim 28 wherein the first and secondchambers are adapted to be coupled together to form a sedimentationassembly which is adapted to be placed in a holder and subjected tocentrifugation.
 32. The method of claim 28 wherein the first chamberreceives the suspension from a system for isolating cells.
 33. Themethod of claim 28 wherein the suspension includes stem cells that havebeen isolated according to the presence or absence of one or moreselected cell markers.
 34. The method of claim 28 wherein the furtherdestination or source contains a solution for washing the cells withinthe suspension.
 35. The method of claim 28 wherein the furtherdestination or source contains a solution for treating the cells withinthe suspension. 36.-52. (canceled)
 53. The method of claim 24 in whichthe sedimentation force field is a centrifugal force field.
 54. Themethod of claim 24 in which the first and second chambers are adapted tobe coupled together to form a sedimentation assembly which is adapted tobe placed in a holder and subjected to centrifugation.
 55. The method ofclaim 24 in which the first chamber receives the suspension from asystem for isolating cells.
 56. The method of claim 24 in which the cellpopulation has been isolated according to the presence or absence of oneor more selected cell markers.
 57. A method of concentrating or washingcells in a suspension, comprising: a. collecting a suspension includinga cell population within a first chamber including a cell concentrationzone; b. sedimenting the cell population to obtain a concentrated cellsuspension within the cell concentration zone of the first chamber; c.flowing the concentrated cell suspension into a second chamber in fluidflow connection with the first chamber under a sedimentation forcefield; d. disconnecting the second chamber from the first chamber; ande. flowing the concentrated cell suspension from the second chamber intoa further fluid destination or source, the further fluid destination orsource being in fluid flow connection with the second chamber andconfigured to be placed together with the second chamber in asedimentation force field.
 58. The method of claim 57 wherein thesedimentation force field is a centrifugal force field.
 59. The methodof claim 57 wherein the first chamber receives the suspension from asystem for isolating cells.
 60. The method of claim 57 wherein thesuspension includes stem cells that have been isolated according to thepresence or absence of one or more selected cell markers.
 61. The methodof claim 57 wherein the further destination or source contains asolution for washing or treating the cells within the suspension.